With the emergence of drug-resistant strains and the cumulative toxicities associated with current therapies, demand remains for new inhibitors of HIV-1 replication. The inhibition of HIV-1 entry is an attractive therapeutic approach with broad implications for HIV/AIDS treatment regimens. Combining computer-aided drug design techniques, based upon novel force field representations of compounds, with medicinal chemistry, we have designed and synthesized novel inhibitors of HIV-1 entry, which represent 5 new distinct core chemotypes for the HIV-1 entry inhibitor class. These compounds have low toxicity, nanomolar potency (0.6-100 nM), and are specific to HIV-1. Moreover, we have identified a compound with improved metabolic stability over the current best-in- class entry inhibitor in development. In this study, we propose to further develop these novel entry inhibitor compounds to potencies that are clinically relevant and improve their ADME/PK profiles, whilst also using them as molecular tools to dissect the biology of HIV-1 entry into host cells. The goals of the proposed studies will be accomplished by three discrete but highly integrated specific aims. In Aim 1, the leads will be optimized through in silico approaches, including bioisosteric replacement and prediction of drug-like qualities, coupled to medicinal chemistry. In Aim 2, the antiviral potency, breadth, and specificity of the analogues will be assessed. Evaluation of their toxicity will be performed in parallel. ADME and PK will be assessed via in vitro and in vivo methodologies, and the efficacy will be determined in the humanized bone-liver-thymus (BLT) mouse model of HIV-1 infection. In Aim 3, we will perform detailed mechanism-of-action studies, generate resistance mutants, and investigate the binding site of the compounds using complementary structural techniques including X-ray crystallography, cryo-EM, and negative-stain single-particle electron microscopy. The data from these studies will be used in the future development of next-generation Env-targeted inhibitors with high genetic barriers and unique modes of action. Such compounds will also serve as molecular probes for HIV-1 Env functions, providing novel insight into the complex series of structural transitions through which the Env molecular machine proceeds, in addition to illuminating the details of an elusive site of vulnerability.